Gelred 1

The PCR amplifications were performed in a MJ Mini^TM Gradient Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA) in a total reaction volume of 25 µL containing 2 µL of template DNA (40 ng), 1× buffer (67 mM of Tris-HCl (pH 8.8), 16 mM of (NH₄)₂SO₄, 0.1% of Tween 20), 200 μM of each dNTP (Grisp, Porto, Portugal), 1.0 U of SuperHot Taq DNA Polymerase (Genaxxon Bioscience, Ulm, Germany), 3.0 mM or 1.5 mM of MgCl₂ for 916/916-R and 18SRG-F/18SRG-R primers, respectively, and 200 nM or 240 nM of each primer, 916/916-R and 18SRG-F/18SRG-R, respectively (Table 1). The amplification programs were defined as follows: initial denaturation at 95 °C for 5 min; 40 cycles (for primers 916/916-R) or 33 (18SRG-F/18SRG-R) at 95 °C for 30 s, 56 °C (916/916-R) or 65 °C (18SRG-F/18SRG-R) for 30 s and 72 °C for 30 s; and a final extension at 72 °C for 5 min.
PCR products were verified by electrophoresis in a 1.5% agarose gel stained with GelRed 1× (Biotium, Inc., Hayward, CA, USA) and carried out in 1× SGTB (Grisp, Porto, Portugal) for 25 to 30 min at 200 V. Agarose gel visualization was performed in a UV light tray Gel Doc™ EZ System (Bio-Rad Laboratories, Hercules, CA, USA), recording a digital image with Image Lab software version 5.2.1 (Bio-Rad Laboratories, Hercules, CA, USA). Each extract was amplified at least in two independent runs.

The amplificability of DNA (i.e. monitoring for PCR inhibitors) was verified using a plant-specific primer pair VPRBCP1/VPRBCP2 targeting the ribulose 1,5-diphosphate carboxylase/ oxygenase gene (rbcL) of the plant chloroplast, as described by Mbongolo Mbella et al. [11 (link)]. The reaction was set up in a volume of 25 μl with 1× PCR buffer (pH 8.3), 200 μM of dNTP mix, 1.5 mM MgCl₂, 0.24 μM of each primer, 1.5 U of Platinum® Taq DNA polymerase (Thermo Scientific, Waltham, USA) and 2 μl DNA sample, in a GeneAmp PCR System 2400 thermocycler (Applied Biosystems, Foster City, USA), as follows: 95 °C for 3 min, 40 cycles at 94 °C for 1 min, 60 °C for 1 min, and 72 °C for 1 min with an additional extension at 72 °C for 7 min. Primers were synthesized and purified by Invitrogen (Thermo Scientific). The PCR products were loaded in a 2% (w/v) agarose gel and stained with GelRed 1× (Biotium, Fremont, USA).

PCR amplifications were carried out in a total reaction volume of 25 μL, containing 2 μL of DNA extract (10 ng), 67 mM Tris-HCl (pH 8.8), 16 mM of (NH₄)₂SO₄, 0.1% of Tween 20, 200 µM of each dNTP, 1.0 U of SuperHot Taq DNA Polymerase (Genaxxon Bioscience GmbH, Ulm, Germany), 2.0 mM of MgCl₂, 200 nM (ITS2A-F/ITS2A-R, matKO-F/matKO-R) or 280 nM (LE1/LE2, EG-F/EG-R) of each primer (Table 1). The reactions were performed in a MJ Mini™ Gradient Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA) using the following programs: (i) initial denaturation at 95 °C for 5 min; (ii) 35 cycles at 95 °C for 30 s, 62 °C (ITS2A-F/ITS2A-R, matKO-F/matKO-R) or 60 °C (LE1/LE2) or 63 °C (EG-F/EG-R) for 30 s and 72 °C for 30 s; (iii) final extension at 72 °C for 5 min. Each extract was amplified at least in duplicate assays.
Electrophoresis was carried out in a 1.5% agarose gel containing Gel Red 1× (Biotium, Hayward, CA, USA) for staining and SGTB 1× (GRiSP, Research Solutions, Porto, Portugal) was used to confirm amplicons. Agarose gels were visualized under a UV light tray Gel Doc™ EZ System (Bio-Rad Laboratories, Hercules, CA, USA) and a digital image was obtained with Image Lab software version 5.2.1 (Bio-Rad Laboratories, Hercules, CA, USA).